In singleplex assays an analyte is the chemical component measured in an analytical procedure. In immunoassay, the analyte is either an antibody or an antigen. Antibodies are proteins in the blood that are produced by the immune system for protection against foreign bodies, while the foreign bodies are the antigens. The antibodies bind to the antigens. The antigens or the antibodies are labelled before analysis, in order to give a measurable signal. This label can be an enzyme, a radioactive isotope, or fluorescein. The signals obtained from an immunoassay can be radioactivity or emission of light. These signals are commonly called responses. The immunoassay involves chemical reactions between clinical samples obtained from patients and reagents (i.e. chemical solutions) performed under standardised conditions. The result is a response that is related to the concentration of the analyte in the sample. In competitive immunoassay, the analyte is unlabelled and competes with labelled molecules. The response is then a decreasing function of the analyte concentration. In noncompetitive immunoassay the labelled molecules bind to the analyte, and the response is an increasing function. In either case, the exact relationship between response and concentration needs to be estimated. This estimation is called calibration. For calibration, samples with known concentrations are required. These specific samples are called calibrators or standards, and are usually prepared in advance. For example, a single sample with a known high concentration can be dissolved in water or animal serum to produce calibrators with a few specified lower concentrations covering the range of measurement. When discussing statistical design for calibration, the specified calibrator concentrations are called design points. Because the calibrators are specially prepared, but the samples are not, the calibrators and the clinical samples may react in slightly differently ways. Usually, a set of clinical samples with unknown concentrations is assayed together with the calibrators in an assay run. A calibration curve is fitted to the responses of the calibrators. This curve can be a straight line or some other monotonic function. The responses of the clinical samples are transformed into estimates of concentration through the fitted calibration curve. This method for estimation of sample concentrations is called inverse prediction.
Because the relationship between response and concentration may change from one assay run to another, calibrators are often included in each assay run, so that each can be calibrated separately. However, in some systems it is assumed that the relationship is stable, so that calibration needs to be performed less often, for example only once a month or when new batches of reagents are taken into use (Forkman J., Doctoral Thesis, Swedish University of Agricultural Sciences, Uppsala, 2008, ISSN 1652-6880, ISBN 978-91-86195-13-7).
Multiplex assays, by which analytes of multiple specificities are detected in a single sample specimen using a single reaction mixture of reagents, are known in the art. An important component of these assays is the calibration system used to define the level of reagent i.e. antibody or biomarker that is measured by the assay. Classically, these levels were reported using a number of arbitrary units, depending on the degree of quantitation afforded by the assay system. In qualitative assays, the targeted molecule in the serum specimen is reported as positive or negative based on the level of the response signal measured, as compared with a pre assigned positive threshold level. In a number of semi quantitative assays, both a positive/negative result, the magnitude of the signal measured (eg, luminescent units [LU], millivolts [mVolts]), the class score, the adjusted or normalized counts (from modified scoring systems), or the percent of the lowest control (alternative scoring system) are reported. The magnitude of the signal is related in terms of rank order to (but not consistently directly proportional to) the quantity of the molecule present in the test serum.
We will here exemplify with three different test types of multiplex assays known in the art:
1) Analysis of specific IgE;
2) Analysis of specific IgG; and
3) Analysis of non-immunoglobulin biomarkers (antigens).
Common analysis methods for specific immunoglobulins are 1) specific IgE analysis for the purpose of detecting allergy/hypersensitivity, and 2) specific IgG analysis for the purpose of detecting for example autoimmune diseases or infectious diseases. Disease relevant antigens are deposited onto a micro array at defined locations. These antigens are exposed to a patient sample including immunoglobulins that may bind to a selected antigen. The specific immunoglobulin is detected with an immunoglobulin specific reagent (reporter molecule) that interacts with the specific immunoglobulin, and that interaction can be analysed through the detection system. It is thereby possible to detect all different immunoglobulins specific for a certain antigen. For test type 3), analysis of non-immunoglobulin biomarkers (antigens), such as prostate cancer biomarkers in serum, molecules that have a capability to bind to the biomarkers of interest are deposited onto a micro array at defined locations. The deposited molecules may for example be biomarker-specific antibodies, enzymes or other molecules that are complementary to the biomarkers of interest. The deposited molecules are exposed to a patient sample including biomarkers that may bind to a selected deposited molecule. The specific biomarker is detected with a biomarker specific reagent (reporter molecule) that interacts with the specific biomarker, and that interaction can be analysed through the detection system.
For example, in the field of specific IgE detection, WO2002029415 A1 describes a method for the detection of an allergen-specific immunoglobulin in a sample, and a method for in vitro diagnosis of allergies in an individual. Clinical manifestations such as asthma, hay fever, atopic eczema and gastro intestinal symptoms develop after exposure to specific allergens. Determination of the sensitization pattern to specific and/or cross reactive allergen components assists in a more detailed evaluation of the allergic patient.
Commercially available IgE antibody immunoassays can be classified into a qualitative, semi quantitative, or quantitative assay, depending on the degree to which the assay result accurately reflects the quantity of IgE antibody in the test specimen and the assay's precision requirements. Such immunoassays traditionally measure either the total serum IgE levels or allergen-specific IgE levels.
However, while different technology platforms report their IgE results in seemingly identical classes or units, studies have shown differences between technology platforms in the ability to detect total IgE and specific IgE activity (Wood R A et al, Ann Allergy Asthma Immunol. 2007, 99: 34-41).
Quantitative IgE antibody assays employ the most advanced methods of assay calibration. The purpose of the calibration portion of the quantitative assay is to define the dose-response relationship of the assay so response results obtained by testing patients' sera can be interpolated in dose units that relate to the relative quantity of IgE antibody in the serum. Both homologous and heterologous interpolation methods have been successfully used. The homologous interpolation procedure promotes overall assay parallelism and maximizes the assay's working range by using the same solid-phase allergen throughout the assay, and constructing a calibration curve with human IgE antibody of the same allergen specificity as is to be detected in the test sera. In general, the IgE antibody-containing reference serum pool dilutes out in the same manner as the test serum IgE, thus ensuring assay parallelism. The primary limitation of this approach is the requirement for liter quantities of human serum pools that contain IgE antibody specific for each allergen specificity to be tested. It is difficult to maintain a serum bank that can supply these large quantities of human serum in a reproducible manner between lots, especially for the less common allergen specificities. Because of constraints placed on assays using the homologous interpolation calibration as a result of limited IgE antibody-containing human serum pools, heterologous interpolation from a total IgE calibration curve has been adopted as the calibration strategy for present day quantitative IgE antibody assays that involve hundreds of different allergen specificities. The heterologous interpolation system has become the industry standard. In the heterologous interpolation system, a total serum IgE calibration curve is run simultaneously with the allergen-specific IgE portion of the assay, using an IgE calibrator that is traceable to the WHO 75/502 Standard (I/LA20-A2 Analytical performance characteristics and clinical utility of immunological assays for human immunoglobulin E (IgE) antibodies and defined allergen specificities; Approved Guideline, ISBN no. 1-56238-695-6).
IMMUNOCAP ISAC® is an in vitro diagnostic test using microarray chip technology. It allows simultaneous measurement of specific molecules in a single test, using only a few μl of fluid, e.g. serum or plasma sample. It may be used for analysis of any biomarker, including IgE, IgG and non-immunoglobulin biomarkers.
For example, in the case of analysing specific IgE antibodies by use of IMMUNOCAP ISAC®, a specific IgE (sIgE) chip delivers results for over a hundred components from more than 50 allergen sources. Allergen components that are immobilized on a solid substrate in a microarray format react with the specific IgE in the patient sample. After washing away nonspecific IgE, fluorescence-labeled anti-human IgE antibody is added to form a complex. After incubation, unbound fluorescence labeled anti-human IgE antibodies are removed by washing. The procedure is followed by fluorescence measurement using an appropriate microarray scanner. The higher the response value, the more specific IgE is present in the specimen.
The test results are analyzed with PHADIA® Microarray Image Analysis (MIA) Software and ISAC Standardized Units for specific IgE (ISU-E) are calculated (Protein microarrays for the diagnosis of allergic diseases: state-of-the-art and future development, Clinical Chemical Laboratory Medicine, Volume 43, Issue 12, Pages 1321-1326).
The results are presented semi-quantitatively in four classes (0=Undetectable or Very Low, 1=Low, 2=Moderate to High, 3=Very High). Phadia MIA Software automatically performs this calculation.
Calibration of an IMMUNOCAP ISAC® microarray chip is made against an in-house reference preparation, or calibration reagent, and measured IgE antibody concentrations are expressed as arbitrary units; ISAC Standardized Units for IgE (ISU-E). The IMMUNOCAP ISAC® in-house reference preparation is calibrated against IMMUNOCAP® Specific IgE (with antibody concentrations expressed as kilo-unit IgE per liter; kUA/l), which is standardised against the WHO reference preparation 75/502 for IgE (Hamilton R G, Assessment of human allergic diseases. In: Clinical Immunology, Principles and Practice, ed. Rich R R, 3rd ed, 2008, p. 1471-84; see page 1476).
IMMUNOCAP ISAC® may also be used in a similar way to analyse specific IgG and/or other biomarkers (antigens and antibodies).
The present calibration systems normally include the independent calibration of each antigen towards the corresponding specific antibody. This may be illustrated by Biorad's Bioplex ANA screen, which uses multiplex immunoassay flow, and which detects the presence of clinically relevant circulating autoantibodies in serum or plasma. At the same time, this is an example of the second type of multiplex assays as mentioned above, i.e. the analysis of specific IgG. The Bioplex system uses a bead based multiplex assay format and the calibration process is described as follows: “While the identity of the dyed beads is determined by the fluorescence of the dyes, the amount of antibody captured by the antigen is determined by the fluorescence of the attached PE” (i.e. phycoerythrin; the fluorescent detection molecule). “Raw data is calculated in relative fluorescence intensity (RFI) and fluorescence ratio (FR). Three additional dyed beads, Internal Standard Bead (ISB), Serum Verification Bead (SVB) and a Blank Bead (BB) are present in each reaction mixture to verify detector response, the addition of serum or plasma to the reaction vessel and the absence of significant non-specific binding in serum or plasma. Refer to the BioPlex 2200 System Operation Manual for more information. The instrument is calibrated using a set of six (6) distinct calibrator vials, supplied separately by Bio Rad Laboratories. For dsDNA, six (6) vials, representing six (6) different levels of antibody concentrations, are used for quantitative calibration, and results for patient samples are expressed in IU/mL. Results of sA IU/mL are negative, 5-9 IU/mL are indeterminate, and results of 10 IU/mL or higher are considered positive for dsDNA antibody. For the other twelve (12) beads, four (4) vials representing four (4) different antibody concentrations are used for semi-quantitative calibration. The result for each of these antibodies is expressed as an antibody index (AI). An AI of 1.0 indicates an antibody cut-off concentration that corresponds to approximately the 99th percentile of values obtained from a non-diseased population; results of 1.0 or higher are reported as positive. Results of <1.0 are reported as negative” (Biorad, Bioplex 2200 Ana Screen SIO(k) Summary, FDA 510(k), SIO(k) Number k041658).
The third type of multiplex assays includes the analysis of non-immunoglobulin biomarkers (antigens), e.g. prostate cancer biomarkers. This is an example in which a traditional singleplex immunoassay is converted into a multiplex format. This has been exemplified through several bead based assays as well as limited multiplexing using various solid arrays. In all these assays it is of essence that each individual test is calibrated separately, which tends to become tedious and cumbersome when running multiplex formats.
Beckman Coulter describes the calibration of its Access Hybritech free PSA assay, which is an analysis of the free form of the prostate cancer biomarker PSA, as a set of 5 different standard points and one negative sample totaling 6 different calibration intervals. It is also evident that the free PSA concentrations are dependent on the standard used to calibrate the assay (Beckman Coulter, Inc., 2010, A85087C, Access Hybritech free PSA).
At present, as described above, calibration of immunoassays for the detection of different types of molecules usually necessitates running several calibration samples for each test, including calibration samples of different concentrations and calibration samples containing different calibrator molecules. Consequently, such a calibration technique is time-consuming and may be imprecise due to systematic assay system variability over time.
The object of the present invention is to provide a reference preparation or calibration reagent that eliminates or at least reduces the above-mentioned problems connected to the techniques presently known.